Title of Invention

A PROCESS FOR BIOLOGICAL PRODUCTION OF HYDROGEN.

Abstract A bioprocess for producing hydrogen gas at high rate and yield and in particular to a bio-process for producing hydrogen using a selective microbial strain Enterobacter cloacae 1IT - BT 08.The process basically involves subjecting immobilizing Enterobacter Cloacae IIT-BT-08 to bio reaction on environmental friendly solid matrices and thereby obtaining the hydrogen. The above process is environmentally friendly as lignocellulosic agro-residues are used as support matrix for generation of pollution free gaseous field. The process is an efficient biological hydrogen production process which would favour high rate/yield of continuous hydrogen production, would be environment friendly and favour generation of pollution free gaseous fuel. The improved bio-process would not have the problem of reversion effect even in the absence of selective process and advantageously does not involve the use of hazardous chemicals and is also not energy intensive.
Full Text The present invention relates to a bioprocess for producing hydrogen gas at high rate and yield and in particular to a bio-process for producing hydrogen using a selective microbial strain Enterobacter cloacae IIT - BT 08.
At present hydrogen is produced mainly from fossil fuels, biomass and water.
The methods of hydrogen production from fossil fuels are :
1. Steam reforming of natural gas,
2. Thermal cracking of natural gas,
3. Partial oxidation of heavier than naphtha hydrocarbons,
4. Coal gassiflcation,
The methods of hydrogen production from biomass are :
1. Pyrolysis or gassification (which produces a mixture of gases, i.e. (H2 ,CH4, CO2 , CO,N2).
The methods of hydrogen production from water are :
1. Electrolysis,
2. Photolysis,
3. Thermochemical process,
4. Direct thermal decomposition or thermolysis,
5. Biological production,
Out of the above listed processes, nearly 90% of hydrogen is produced by the reactions of natural gas or light oil fractions with steam at high temperatures (steam reforming). Coal gasification and electrolysis of water are other industrial methods for hydrogen production. These industrial methods mainly consume fossil fuel as energy source, and sometimes hydroelectricity. However, both thermochemical and electrochemical hydrogen generation processes are energy intensive and not always environmentally friendly. On the other hand, biological hydrogen production processes are mostly operated at ambient temperatures and pressures, thus less energy intensive. These processes are not only environmentally friendly, but also they lead to open a new avenue for the utilization of renewable energy resources which are inexhaustible. In addition, they can also use various waste materials, which facilitates waste recycling.

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Biological hydrogen production processes can be classified as follows:
1. Biophotolysis of water using algae and cyanobacteria;
2. Photodecomposition of organic compounds by photosynthetic bacteria;
3. Fermentative hydrogen production from organic compounds; and
4. Hybrid systems using photosynthetic and fermentative bacteria.
1. Biophotolysis of water using algae and cyanobacteria
This method uses the same processes found in plants and algal photosynthesis, but adapts them for the generation of hydrogen gas instead of carbon containing biomass. Photosynthesis involves the absorption of light by two distinct photosynthetic systems operating in series: a water splitting and O2 evolving system ("photosystem II" or PSII) and a second photosystem (PSI), which generates the reductant used for CO2 reduction. In this coupled process two photons (one per photosystem) are used for each electron removed from water and used in CO2 reduction or H2 formation. In green plants only CO2 reduction takes place, as the enzymes that catalyze hydrogen formation, the hydrogenases, are absent. Microalgae, both eucaryotic (such as the green algae) and procaryotes (the cyanobacteria or blue-green algae), have hydrogenase enzymes, and can produce hydrogen under certain conditions .However, the use of Green algae require light for hydrogen production. Moreover oxygen can be dangerous for the system. As regards the use of Cynobacteria, uptake hydrogenase enzymes are to be removed to stop the degradation of H2. Also it requires sunlight and the oxygen present has inhibitory effect on nitrogenase. Also it is found that carbon dioxide is present in the gas.
2. Photo-decomposition of organic compounds by photosynthetic bacteria
Phototrophic bacteria are indicated in the current literature as the most promising microbial system for the biological production of hydrogen . The major benefits are noted below:
1. high theoretical conversion yields,
2. lack of O2-evolving activity, which causes problem of O2 inactivation of different
biological systems,
3. ability to use wide spectrum of light, and
4. ability to consume organic substrates derivable from wastes and then, for their
potential to be used in association with wastewater treatment.

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However the problem with such use of photo synthetic bacteria is that it also requires light for the production of hydrogen. The fermented broth will cause water pollution problems. Also it is found that carbon dioxide is present in the gas.
3. Fermentative hydrogen production from organic compounds
Hydrogen evolution by fermentation has been treated with little attention, while hydrogen evolution by photo synthetic microorganisms has been extensively studied. The evolution of hydrogen by fermentation has, however, several advantages for industrial production, such as:
(a) Fermentative bacteria have very high evolution rate of hydrogen,
(b) They can produce hydrogen constantly through day and night from organic
substrates,
(c) They can have growth rate good for supply of microorganisms to the production
system,
Therefore, the fermentative evolution is more advantageous than photochemical evolution for mass production of hydrogen by microorganisms. Fermentative hydrogen production can be maximized through the effective coupling of the following iactors :
(a) An accessible and rich source of electron and biochemical electron pump,
(b) An active hydrogenase, however even such fermentative bacteria is found to suffer
from demerits in that the fermented broth is required to undergo further treatment
before disposal otherwise it will create water pollution problems. Also in such system
the carbon dioxide is found to be present in the gas.
4. Hybrid system using photosynthetic and fermentative bacteria
Hybrid system comprises of non-photo synthetic and photosynthetic bacteria. It can enhance the hydrogen production. Variety of carbohydrates can be digested by C. butyricum. This bacterium produces hydrogen with the degradation of carbohydrates without using light. Resulting organic acids could be sources for photosynthetic bacteria to produce hydrogen. Anaerobic bacteria decompose carbohydrates to obtain both energy and electron. Because reaction only with negative free energy could be possible, organic

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acids formed by the anaerobic digestion could not be decomposed to hydrogen any more. Complete degradation of glucose to hydrogen and carbon dioxide is impossible by anaerobic digestion. Photosynthetic bacteria could use light energy to overcome the positive free energy reaction (bacteria can utilize organic acids for hydrogen production). The combination of the both kinds of bacteria not only reduces the light energy demand of photosynthetic bacteria but also increases hydrogen production.
Immobilized whole cell systems have several advantages as compared to suspended cells systems. One important advantage is to reuse the system repeatedly. Continuous and stable hydrogen production was achieved by immobilizing the combined system of Phormidium valderianum, Halobacterium halobium and Escherichia coli in a PVA-alginate film. The intermittent supply of nitrogen was found to be essential to retain cellular metabolic activities, which in turn showed prolonged production of hydrogen Bagai R, Madamwar D. Prolonged evolution of photohydrogen by intermittent supply of nitrogen using a combined system of Phormidium valderiannum, halobacterium halobium and Escherichia coli. Int J Hydrogen Energy. 1998; 23: 545-550. Substrate like glucose can be utilized by this system and therefore, it may have a potential significance in removing organic materials from the wastewater and simultaneously producing hydrogen. Rhodobacter sphaeroides O.U. 001 was immobilized in calcium alginate beads and this was used for continuous hydrogen production. It enhanced hydrogen production two to three folds. Hydrogen production from the wastewater of a tofu factory was examined by using Rhodobacter sphaeroides immobilized in agar gels. The maximum rate of hydrogen production observed from the wastewater was 2.1 1 h" m"2. Zhu H, Suzuki T, Tsygankov A A, Asada Y, Miyake J. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. . Int J Hydrogen Energy. 1999; 24: 305-310.
However in spite of such advantages in the use of immobilized whole cell systems, it is found that the generation of the hydrogen gas using such known systems was not to the desired extent and thus there remained a need in the art to make such system efficient and cost effective.

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Object of the Invention
It is thus the basic object of the present invention to provide an efficient biological hydrogen production process which would favour high rate of continuous hydrogen production.
Another object is directed to provide an efficient biological hydrogen production process which would be environment friendly and favour generation of pollution free gaseous fuel.
Another object is to provide an improved bio-process for the production of hydrogen which can generate yield generation than previously known conventional bio process.
Yet another object of the present invention is to provide for an improved bio-process for the production of hydrogen which would not have the problem of reversion effect even in the absence of selective process.
Yet further object is to provide for continuous hydrogen production on large scale without any use of hazardous chemicals and which is not energy intensive.
In our copending Patent Application No. 464/Cal/2001 of 21.8.2001, there is disclosed a novel microbial cell Enterobacter Cloacae IIT-BT 08 . In particular, the said application disclosed the characteristics of the microorganism.
It has now been found that the said novel Enterobacter Cloacae IIT-BT 08 is highly efficient for producing pollution free gaseous fuel.
Thus according to the present invention there is provided a process for biological production of hydrogen comprising subjecting immobilizing Enterobacter Cloacae IIT-BT-08 to bio reaction on environmental friendly solid matrices and thereby obtaining the hydrogen. Preferably, in the above process, environmentally friendly lignocellulosic agro-residues are used as support matrix for generation of pollution free gaseous field. It has been found that the appropriate lignocellulosic materials that may be employed for the purpose of the present invention includes rice straw, bagasse, cocnut coir, jute fiber, wood shavings etc., wherein coconut coir was found to provide the best results in terms of cell retention, packing density, concentration of the cells and the rate of hydrogen production.

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In accordance with a preferred aspect, the present invention comprises of
feeding the lignocellulosic agro-residues as support matrix in a bio reactor wherein the
said immobilisation of the selected Enterobacter Cloacae IIT-BT 08 is carried out
maintaining the anaerobic conditions in said bio reaction;
exiting the effluent through a gas exit; and collecting the gas in a gas collector.
In particular, in the above process, to maintain the anaerobic condition in the bio reactor, a liquid trap is operatively connected to the bio reactor such that the over flow of the outgoing liquid from the reactor takes place through this trap.
The gas collection is preferably carried out through water displacement and collected in a cylindrical column. A peristaltic pump can be used to feed the lignocellulosic matrix media from a feed tank to the said immobilizing whole cell bioreactor.
In accordance with another preferred aspect of the process of the invention, the gas generated in the bio reactor is passed through a carbon dioxide absorber to remove the carbon dioxide from the gas prior to its collection.
In accordance with another aspect of the invention, it is found that in order to avoid the problem of gas hold up when using immobilizing whole cell bio reactors, Rhomboid bio reactor with convergent divergent geometry should be preferably used. Importantly, by use of such Rhomboid bioreactor, it was possible to reduce the gas hold ups by 67% in comparison to tubular reactor.
This is basically due to higher turbulence created by convergent divergent geometry of the bio reactor (velocity valid from 4 cm / h to 37 cm / h in the convergent section and from 1.3 cm / h to 12 cm / h those in the divergent section) which was responsible for continuous renewal of the surface and thus the substrate has a better interaction with cells in the bio reactor.
The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations of the process discussed in relation to accompanying Figure 1.

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Description of the Accompanying Drawings
As illustrated in Fig. 1, the Feed tank (1 )is used to store the media that is the environment friendly solid matrices such as lignocellulosic agro residues. From the said feed tank the solid matrices are feed into the immobilized whole cell bio reactor (2) having the immobilised whole cell, Enterobacter cloacae IIT-BT 08.The media is passed at the bottom and outgoing liquid comes out from the top as shown in the figure. The hydrogen generation process is actually carried out in this reactor.
A Liquid trap (3) is used mainly for maintaining anaerobic condition in the bioreactors. Overflow of the outgoing liquid from the reactor takes place through this trap.
In accordance with a preferred aspect a Carbon dioxide absorber (4) is used to remove carbon dioxide from the gas. The carbon dioxide absorber preferably containing 50 % KOH solution. Effluent is outgoing liquid on the reactor.
A gas exit (6) is provided to exit the gas which can be used. A gas collector (7) is provided to collect the gas through displacement of water. Preferably, a peristaltic pump is used to send the media from the feed tank to the immobilized whole cell bioreactor.
Studies were carried out to ascertain the efficacy of the biological process for the production of hydrogen in accordance with the present invention vis-a-vis known biological processes. Preferably, a quasi steady state (5% variation) was confirmed at each dilution rate with respect to constant values of hydrogen evolution rate glucose and cell concentration in the effluent. The experiments were repeated at different flow rate to get maximum hydrogen production and sugar utilization. Anaerobicity was maintained in all experiments. All the bioreactors used in the present studies were operated continuous for a minimum period of 40 days to find out operational stability of the system. The results obtained are detailed in Table 1 hereunder

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9

a = medium contains different components b = expressed as ml H2/g Bchlorophyll-a. h c = expressed in mmol / mg protein, h
As clearly illustrated in Table 1, the bioprocess involving the selective Enterobacter cloacae BT-08 provided for high generation of hydrogen gas vis-a-vis the other organisms tried.
In accordance with a further aspect of the invention the advantages in selective use of the support matrix of lignocellulosic agroresidue in the process of the invention vis-a-vis other solid matrix was ascertained and for the purpose the following studies were carried out.
The present system was compared with other known immobilized system and the results obtained are detailed in the Table 2 hereunder
TABLE 2: Comparative studies on continuous H2 production in a packet-bed reactor using immobilized whole cells


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The results clearly confirm the suitability of the present system. While several lignocellulosic solid matrices were used in the studies, a special type of solid matrix was found suitable for hydrogen production, mainly due to the enhancement of density of the cells in the solid matrices.
In accordance with a further aspect of the invention, the suitability of the bioreactor to carry out the process of the invention was studied. For the purpose different bioreactor configurations were used to overcome the gas hold up problem in the system. The results obtained using various bioreactors are detailed in Table 3.
TABLE 3: Performance of different bioreactor using Lignocellulosic Agroresidues as solid matrix
Volume of each bioreactor : 380 ml

As evident from the details under Table 3 the Rhomboid bioreactor was found most suitable for this system. The maximum rate of hydrogen production was found to be more than two times higher than the batch system.
Importantly the major advantage of this process is that use of environmental friendly solid matrix for the immobilization of the whole cell and development of suitable bioreactor configuration for the high rate of hydrogen production.

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It is thus possible by way of the present invention to provide a novel process for hydrogen production which would achieve a higher rate of hydrogen production than the other known bio-processes.
In the process of the invention the yield of hydrogen is higher than other reported strain. The process provides for recovery of more energy in the form of Hydrogen.
The following Table 4 is provided with respect to the performance of some suitable "lignocellulosic agro-residues" as solid matrix
Table Comparative studies on H2 production using freely suspended and immobilized K.cloacae 11T-BT 08 on different natural biopolymers.
Table 4

Bioreactor configuration: tubular: bioreactor volume: 380 ml; void fraction : 0.65; temperature: 36°C; media: MYG
Rice straw, bagasse and coconut coir were considered as solid matrices and named as SM-A, SM-B and SM-C respectively.

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WE CLAIM
1. A process for biological production of hydrogen comprising subjecting immobilizing
Enterobacter Cloacae IIT-BT-08 to bio reaction on lignocellulosic agro-residues
selected from rice straw, begasse, coconut coir, jute fibre, wood shavings under
anaerobic conditions and thereby obtaining the hydrogen.
2. A process as claimed in claim 1 comprising:
feeding the lignocellulosic agro-residues as support matrix in a bio reactor wherein the said immobilisation of the selected Enterobacter Cloacae 1IT-BT 08 is carried out maintaining the anaerobic conditions in said bio reaction;
exiting the effluent through a gas exit; and collecting the gas in a gas collector.
3. A process as claimed in anyone of claims 1 or 2 wherein a liquid trap is used to
maintain the anaerobic condition in the bio reactorwhich is operatrively connected to
the bio reactor such that the over flow of the outgoing liquid from the reactor takes
place through this trap.
4. A process as claimed in anyone of claims 1 to 3 wherein the gas collection is
preferably carried out through water displacement and collected in a cylindrical
column.
5. A process as claimed in anyone of claims 1 to 4 wherein a peristaltic pump is used to
feed the lignocellulosic matrix media from a feed tank to the said immobilizing whole
cell bioreaclor.
6. A process as claimed in anyone of claims 1 to 5 comprising passing the gas
generated in the bio reactor through a carbon dioxide absorber to remove the carbon
dioxide from the gas prior to its collection.
7. A process as claimed in anyone of claims 1 to 6 wherein a Rhomboid bio reactor with
convergent divergent geometry is used.
8. A process as claimed in claim 7 wherein said bioreactor creates a higher turbulancc
convergent divergent geometry of the bio reactor velocity varied from 4 cm / h to 37.

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cm / h in the convergent section and from 1.3 cm / h to 12 cm / h those in the divergent section .
9. A process as claimed in anyone of claims 1 to8 wherein said solid
matrices from the said feed tank are feed into the immobilized
whole cell bio reactor having the immobilized whole cell
Enterobacter cloacae IIT-BT 08, said media being passed at the
bottom and outgoing liquid comes out from the top .
10. A process for biological production of hydrogen substantially as
described herein and illustrated with reference to the exemplary
illustrations.
A bioprocess for producing hydrogen gas at high rate and yield and in particular to a bio-process for producing hydrogen using a selective microbial strain Enterobacter cloacae 1IT - BT 08.The process basically involves subjecting immobilizing Enterobacter Cloacae IIT-BT-08 to bio reaction on environmental friendly solid matrices and thereby obtaining the hydrogen. The above process is environmentally friendly as lignocellulosic agro-residues are used as support matrix for generation of pollution free gaseous field. The process is an efficient biological hydrogen production process which would favour high rate/yield of continuous hydrogen production, would be environment friendly and favour generation of pollution free gaseous fuel. The improved bio-process would not have the problem of reversion effect even in the absence of selective process and advantageously does not involve the use of hazardous chemicals and is also not energy intensive.

Documents:

00665-cal-2001 abstract.pdf

00665-cal-2001 claims.pdf

00665-cal-2001 correspondence.pdf

00665-cal-2001 description(complete).pdf

00665-cal-2001 drawings.pdf

00665-cal-2001 form-1.pdf

00665-cal-2001 form-18.pdf

00665-cal-2001 form-2.pdf

00665-cal-2001 form-3.pdf

00665-cal-2001 letters patent.pdf

00665-cal-2001 p.a.pdf

00665-cal-2001 reply f.e.r.pdf

665-CAL-2001-FORM-27.pdf

665-cal-2001-granted-abstract.pdf

665-cal-2001-granted-claims.pdf

665-cal-2001-granted-correspondence.pdf

665-cal-2001-granted-description (complete).pdf

665-cal-2001-granted-drawings.pdf

665-cal-2001-granted-form 1.pdf

665-cal-2001-granted-form 18.pdf

665-cal-2001-granted-form 2.pdf

665-cal-2001-granted-form 3.pdf

665-cal-2001-granted-letter patent.pdf

665-cal-2001-granted-pa.pdf

665-cal-2001-granted-reply to examination report.pdf

665-cal-2001-granted-specification.pdf


Patent Number 212605
Indian Patent Application Number 665/CAL/2001
PG Journal Number 49/2007
Publication Date 07-Dec-2007
Grant Date 04-Dec-2007
Date of Filing 04-Dec-2001
Name of Patentee INDIAN INSTITUTE OF TECHNOLOGY
Applicant Address PIN 721302, KHARAGPUR, WEST BENGAL, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 DR.DEBABRATA DAS. DEPARTMENT OF BIOTECHNOLOGY, INDIAN INSTITUTE OF TECHNOLOGY,KHARAGPUR, PIN 721302,WEST BENGAL, INDIA.
PCT International Classification Number N/A
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA